Spatial Compartmentalization Specializes the Function of Aurora A

Spatial Compartmentalization Specializes the Function ofAurora A and Aurora B*□S

Received for publication, March 17, 2015, and in revised form, May 15, 2015 Published, JBC Papers in Press, May 18, 2015, DOI 10.1074/jbc.M115.652453

Si Li‡§, Zhaoxuan Deng§, Jingyan Fu§, Caiyue Xu§, Guangwei Xin§, Zhige Wu§, Jia Luo§, Gang Wang§, Shuli Zhang§,Boyan Zhang§, Fangdong Zou‡1, Qing Jiang§2, and Chuanmao Zhang§3

From the ‡Ministry of Education Key Laboratory of Bio-resources and Eco-environment, College of Life Sciences, Sichuan University,Chengdu, Sichuan 610064 and §Ministry of Education Key Laboratory of Cell Proliferation and Differentiation and State KeyLaboratory of Biomembrane and Membrane Biotechnology, College of Life Sciences, Peking University, Beijing 100871, China

Background: Aurora kinases show different localizations and play distinct roles, yet the mechanisms remain largelyunknown.Results: Different localization leads to functional divergence of the Auroras and their N termini also contribute to thelocalization.Conclusion: Both N/C termini of Aurora A/B contribute to their spatial compartmentalization and function.Significance: Functional divergence of Aurora kinases is largely determined by their localizations through binding withpartners/substrates.

Aurora kinase A and B share great similarity in sequences,structures, and phosphorylation motif, yet they show differentlocalizations and play distinct crucial roles. The factors thatdetermine such differences are largely unknown. Here we tar-geted Aurora A to the localization of Aurora B and found thatAurora A phosphorylates the substrate of Aurora B and substi-tutes its function in spindle checkpoint. In return, the centro-some targeting of Aurora B substitutes the function of Aurora Ain the mitotic entry. Expressing the chimera proteins of theAuroras with exchanged N termini in cells indicates that thedivergent N termini are also important for their spatiotemporallocalizations and functions. Collectively, we demonstrate thatfunctional divergence of Aurora kinases is determined by spatialcompartmentalization, and their divergent N termini also con-tribute to their spatial and functional differentiation.

The Aurora kinases belong to the serine/threonine kinasefamilies and are essential for the cell cycle control in theeukaryotes (1, 2). Many low species have only one Aurora,whereas the mammals have at least three, Aurora A, B, and C,among which Aurora C resembles Aurora B but regulates mei-osis and mitosis during early development (2– 4). Aurora A andB display different localizations and functions during the cellcycle. Aurora A is located to the centrosomes throughout thecell cycle, spreads on the spindle microtubules in metaphase,and is relocated to the central spindle in anaphase and telo-

phase (5– 8). In contrast, Aurora B is mainly located on thecentromere in early mitosis, where it serves as a component ofthe chromosome passenger complex, and on the midzone/mid-body during the cytokinesis (3). In accordance with the spatio-temporal localization divergence, Aurora A and B performdistinct functions. Aurora A is required for centrosome matu-ration, mitotic entry, and centrosome separation, whereasAurora B mainly regulates spindle assembly checkpoint, kine-tochore attachment, and cytokinesis (3, 9 –11).

Both molecules of Aurora A and B have a divergent N termi-nus and a conserved catalytic domain-containing C terminus insequence (12–14). Aurora B combines with INCENP, Survivin,and Borealin to form the chromosome passenger complex,which is required for activation and specialized localization ofAurora B (3). Aurora A interacts with a microtubule-associatedprotein TPX2 that not only regulates the localization but alsothe activation of Aurora A on the spindle in prometaphase andmetaphase (15). Aurora A also interacts with a variety of bind-ing partners/substrates including centrosome-localized Ajuba,Bora, and PAK1 and functions on the centrosomes (10, 16, 17).

In the known cases of yeast (Saccharomyces cerevisiae andSchizosaccharomyces pombe), mycetozoa (Dictyostelium dis-coideum), primitive deuterostome (starfish, ascidian, andurchin), and so on, the only Aurora kinase shows the localiza-tion and function of both Aurora A and B of high species (18 –20). Phylogenetic analysis shows that both Aurora A and B arelikely evolved from a primitive bifunctional Aurora kinase,although how the differentiation of their Auroras is formedremains largely unknown (21). It is well accepted that duplica-tion and divergence are the primary means by which new pro-teins and pathways are created. There are many factors includ-ing the substrate specificity, the specified binding of proteins,and the spatial mechanisms like the scaffolds, the regulation ofreactions, and the formation of macromolecular complexesthat contribute to the evolution and divergence of the proteins(22, 23). Generally, fully conserved positions of proteins mayconfer the proteins’ common functional features, whereas less

* This work was supported by State Key Basic Research and DevelopmentPlan of the Ministry of Science and Technology of China Grants2010CB833705 and 2014CB138402 and the National Natural ScienceFoundation of China Grants 31430051, 31371365, 91313302, and31030044. The authors declare that they have no conflicts of interest withthe contents of this article.

□S This article contains supplemental Movies S1–S8.1 To whom correspondence may be addressed. E-mail: [email protected] To whom correspondence may be addressed. E-mail: jiangqing@pku.

edu.cn.3 To whom correspondence may be addressed. E-mail: zhangcm@pku.

edu.cn.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 28, pp. 17546 –17558, July 10, 2015© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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conserved specificity-determining positions are related to theirdivergent functions. It has been found that both Aurora A and Bmay have similar optimal phosphorylation motifs (24) and thatthey may share some common substrates such as CENP-E,MCAK, Kif2, and RASSF1A on the spindles in cells (25–29).However, the paradox is why Aurora A and B show very differ-ent localizations and play distinct roles during the cell cyclewith such a similarity in sequence and phosphorylation motifaffinity.

In this study we set out to determine how Aurora A and B arespatially compartmentalized for their functional divergence.We found that, in addition to the catalytic conservative C ter-mini, the divergent N termini also regulate their localization,and the resultant spatial compartmentalization further leads tofunctional divergence.

Experimental Procedures

Molecular Cloning—For construction of fusion proteins,GFP-H2B-Aurora A, GFP-CENPB-Aurora A, GFP-Hec1-Au-rora A, GFP-PLK4-Aurora B, wild-type Aurora A, or Aurora Bwas inserted into pEGFP-C1 plasmid. Full-length H2B,CENP-B aa4 1–158 centromere targeting sequence, and full-length Hec1 were cloned by RT-PCR from HeLa cell lysate andinserted to the N terminus of Aurora A sequence. PLK4 aa570 – 820 centrosome-targeting sequence was inserted in theN-terminus of Aurora B sequence. Kinase dead mutation wasgenerated by point mutation PCR. N-terminal and C-terminaltruncation mutant of Aurora A and Aurora B was cloned topEGFP-C1 plasmid. And fusion proteins GFP-Aurora A-B andGFP-Aurora B-A were generated as in Fig. 1 by overlappingPCR.

Cell Culture and Drug Treatment—HeLa cells were culturedat 37 °C under 5% CO2 in DMEM supplemented with 10% fetalbovine serum. To synchronize HeLa cell to mitosis, the cellcycle was blocked by double thymidine treatment to G1-S phaseand then released for 10 h. To generate mitosis cells with mis-aligned chromosomes, HeLa was released from single thymi-dine block and then treated with S-trityl-L-cysteine (STLC) for10 h. Kinase activity of Aurora A is inhibited by 50 nM

MLN8237 (Alisertib). Kinase activity of Aurora B is inhibited by200 nM AZD1152.

Immunofluorescence Microscopy—Cells grown on coverslipswere fixed with precooled methanol for 5 min. Then the cover-slips were incubated with primary antibody diluted in 3% BSAovernight at 4°C. After washing 3 times in PBS, coverslips wereincubated with secondary antibody diluted in 3% BSA for 1 h atroom temperature. The coverslips were further washed in PBSand mounted with Mowiol added with 1 mg/ml DAPI for DNAstaining. The antibodies used in immunofluorescence micros-copy include anti-centromere antibody from patients withCREST syndrome (MBL), anti-H3pS10 (Cell Signaling), anti-BubR1 (Abcam), anti-�-tubulin (Sigma), anti-cyclin B1 (SantaCruz).

Western Blotting—Cell lysates in sampling buffer were loadedonto 10% SDS-PAGE gel for electrophoresis and transferred

onto nitrocellulose membranes. The membranes were blockedin TTBS (TBS plus 0.1% Tween 20) containing 1.5% milk for 1 hat room temperature and incubated with primary antibodydiluted in TTBS/milk overnight at 4°C. The membranes werewashed 3 times in TTBS and blocked in TTBS/milk for 1 h,incubated with HRP-conjugated secondary antibody diluted inTTBS/milk for 1 h at room temperature, and then washed 3times. Finally, the blots were developed by chemiluminescence.The antibodies used in Western blotting include anti-phospho-Aurora A/Aurora B/Aurora-C (Cell Signaling), anti-Aurora B(BD Biosciences), anti-Aurora A (produced in our laboratory),and anti-GAPDH (Proteintech).

RNA Interference—For siRNA treatment, duplexed siRNA wereintroduced using Lipofectamine 2000. Cells were processed forimmunofluorescence microscopy 56 h after transfection. siRNAsused include si-Aurora A (5�-GCAGAGAACTGCTACTTAT-3�)and si-Aurora B (5�-GGATCTACTTGATTCTAGA-3�).

Live Cell Imaging—Cells were plated in glass-bottom dishes.RFP-H2B was transfected to indicate the chromosomes. Datawere acquired by a DeltaVision live cell imaging system(Applied Precision) equipped with an Olympus IX-71 invertedmicro-scope and a 60�/1.42 oil objective.

Statistical Analysis—Each data point represents three inde-pendent experiments, and error bars indicate S.D. The statisti-cal significance of differences was calculated with a two-tailedStudent’s t test. Differences were considered significant at p �0.05. *, **, and *** indicate p � 0.05, p � 0.01, and p � 0.001,respectively.

Results

Chromatin-localized Aurora A Phosphorylates Histone H3 inVivo—Because Aurora A and B have common substrates andfunctions on the spindle (30) and some of Aurora B substratesincluding histone H3, INCENP, and Survivin can be phosphor-ylated by Aurora A in vitro (31), we set out to test whetherspecial functions of Aurora A and B are determined by theirdistinct localizations. We speculated that, if the functionaldivergence of Aurora A and B is achieved by their spatial com-partmentalization through specifically binding their substratesand binding partners, the forced localization exchange of theboth would substitute the functions of each other. By fusingAurora A with either histone H2B or the centromere proteintruncate CENP-B1–158 tagged with GFP (GFP-H2B-Aurora Aand GFP-CENPB-Aurora A) and transiently expressing thesefusion proteins in cells, we found that the fusion protein GFP-CENPB-Aurora A was localized to the nucleus and primarilythe centromere during the cell cycle, and a fraction of it wasrelocated to the spindle/poles as did the wild-type Aurora Afrom prophase to metaphase and that the fusion protein GFP-H2B-Aurora A was located primarily on the chromatin/chro-mosomes during the cell cycle (Fig. 1A and supplemental Mov-ies S1 and S2). As it is known that Aurora B is localized ininterphase nucleus and relocated to the centromere in earlymitosis, we concluded that GFP-H2B-Aurora A and GFP-CENPB-Aurora A proteins had been localized to the areas towhich Aurora B is preferentially localized. By probing the activephosphorylation status of Aurora A at Ser-232 using a phos-pho-specific antibody, we also found that these two fusion pro-

4 The abbreviations used are: aa, amino acid(s); STLC, S-trityl-L-cysteine; CTS,centrosome targeting sequence; RFP, red fluorescent protein.

Localization Defines Function of Aurora A and B

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teins were also activated on their T-loops like the wild-typeAurora A (data not shown). To evaluate whether these fusionproteins may substitute the functions of Aurora B, we elimi-nated the kinase activity of endogenous Aurora B by treatingHeLa cells with a serial concentration of an Aurora B-specificinhibitor AZD1152. The inhibition efficiency was tested bydetecting the active phosphorylation status of Aurora proteinsusing the phospho-specific antibody. The result showed that, inthe presence of AZD1152 at the concentration of 200 nM andabove, the kinase activity of Aurora B was totally inhibited,whereas the kinase activity of GFP-CENPB-Aurora A was lessaffected by AZD1152 (Fig. 1B). Then the cells expressing GFP-

H2B-Aurora A and GFP-CENPB-Aurora A were treated with200 nM AZD1152 for 1 h and processed for immunofluores-cence microscopy. The result showed that the Aurora B sub-strate histone H3 Ser-10 was extensively phosphorylated inalmost all transfected cells by GFP-H2B-Aurora A regardlessthe period of the cell cycle (96.52%). In contrast, only a verysmall amount of cells transfected with GFP-Aurora A showedslight phosphorylation of histone H3 Ser-10. Moreover, in cellsexpressing GFP-CENPB-Aurora A that was located at the cen-tromere, the phosphorylated histone H3 Ser-10 was onlyrestricted at the centromere (Fig. 1, C and D). On the contrary,the kinase dead mutants of GFP-H2B-Aurora A and GFP-

FIGURE 1. Chromatin-localized Aurora A phosphorylates the Aurora B substrate histone H3 in vivo. A, immunofluorescence labeling of HeLa cellstransfected with GFP-Aurora A (AurA), GFP-CENPB-Aurora A, or GFP-H2B-Aurora A with a CREST antibody. The DNA was stained by DAPI. Note that the wild-typeAurora A was localized to the centrosome in interphase, whereas GFP-CENPB-Aurora A was situated to the centromere and GFP-H2B-Aurora A to the chroma-tin/chromosome both in interphase and mitosis. Scale bar, 10 �m. B, HeLa cells transfected with GFP-CENPB-Aurora A were synchronized to prometaphase andtreated with different concentrations of Aurora B inhibitor AZD1152 for 1 h. Cell extracts were subjected to Western blot analysis with antibodies to phospho-Aurora A/-B/-C, Aurora B (AurB) and Aurora A. Note that AZD1152 at 200 nM and above totally inhibited the activation of Aurora B and Aurora C (AurC) indicatedby autophosphorylation, whereas 200 nM AZD1152 did not inhibit the activation of GFP-CENPB-Aurora A. C, histone H3 Ser(P)-10 immunofluorescence labelingof HeLa cells transfected with GFP-H2B-Aurora A, GFP-H2B-Aurora A-KD, GFP-CENPB-Aurora A, GFP-CENPB-Aurora A-KD, or GFP-Aurora A followed by treat-ment with 200 nM AZD1152 for 1 h. The DNA was stained with DAPI. Note that GFP-H2B-Aurora A and GFP-CENPB-Aurora A expression resulted in phosphor-ylation of, and colocalized with histone H3 Ser-10 (H3pS10), whereas GFP-H2B-Aurora A-KD, GFP-CENPB-Aurora A-KD, and GFP-Aurora A did not induce histoneH3 Ser-10 phosphorylation. Scale bar, 10 �m. D, quantitative characterization of HeLa cells treated as in C. Positive staining cells are defined as strongly stainingwith clear edge. Cells with no stain or weak vague staining are not counted as positive staining cells. Bars represent the percentage of the cells with histone H3Ser-10 phosphorylation. Each data point represents 3 independent experiments with each measuring 100 cells, and error bars indicate S.D. E, histone H3Ser(P)-10 immunofluorescence labeling of HeLa cells co-transfected with Aurora B siRNA to knock down endogenous Aurora B and GFP-H2B-Aurora A or RNAiresistant GFP-H2B-Aurora B. Note that both GFP-H2B-Aurora B and GFP-H2B-Aurora A phosphorylated histone H3 Ser-10 (H3pS10). F, quantitative character-ization of HeLa cells treated as in E. Bars represented the percentage of the cells with histone H3 Ser-10 phosphorylation. Each data point represents threeindependent experiments with each measuring 100 cells, and error bars indicate S.D. Scale bar, 10 �m.




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CENPB-Aurora A (GFP-H2B-Aurora A-KD and GFP-CENPB-Aurora A-KD) barely phosphorylated histone H3 Ser-10 (Fig. 1,C and D). Together, these results strongly indicated that his-tone H3 Ser-10 was specifically phosphorylated by GFP-H2B-Aurora A or GFP-CENPB-Aurora A. To further confirmwhether GFP-H2B-Aurora A performed the same function asAurora B, endogenous Aurora B was knocked down in HeLacells by siRNA, and GFP-H2B-Aurora A or GFP-H2B-Aurora Bwas simultaneously expressed. By immunofluorescencemicroscopy, we found that histone H3 at Ser-10 was phosphor-ylated in almost all of the cells expressing GFP-H2B-Aurora Aor GFP-H2B-Aurora B (Fig. 1, E and F). Collectively, theseresults demonstrated that, when forced to locate to the sameplace of Aurora B, Aurora A may phosphorylate the Aurora Bsubstrate histone H3. Thus, we conclude that the specific phos-phorylation of histone H3 Ser-10 by Aurora B rather thanAurora A is indeed caused by the spatial compartmentalizationof the Auroras and that other substrates of Aurora B could alsobe phosphorylated by Aurora A provided that the spatial com-partmentalization of these two Aurora kinases is eliminated.

Kinetochore-targeted Aurora A Performs the Function ofAurora B on the Spindle Checkpoint Regulation—One of themost important functions of Aurora B in mitosis is to regulatethe kinetochore-microtubule attachment and the spindlecheckpoint. The activation of the spindle checkpoint is stim-ulated by Aurora B in two pathways, one of which is thatAurora B destabilizes erroneous kinetochore-microtubuleattachment and indirectly induce spindlecheckpoint.Thecen-tromere-located Aurora B gives rise to a phosphorylation gra-dient around the centromere, and the phosphorylation of theAurora B substrates at the kinetochores depends on their dis-tance from Aurora B at the centromere (32, 33). If microtubulesfrom two opposite poles fail to bind the kinetochores correctly,the tension between the paired kinetochores is low, and AuroraB phosphorylates its multiple substrates of the KMN network(including Hec1, DSN1, Knl1) on the kinetochores, as the dis-tance from the substrates to Aurora B is short and thus activatesthe spindle assembly checkpoint by releasing the microtubulesfrom the kinetochores (34, 35). Another pathway is that AuroraB functions synergically with MPS1 to induce kinetochorelocalization of spindle checkpoint proteins BubR1 and mad2(36, 37). To further explore whether the forced localization ofAurora A on the centromere can substitute Aurora B for thefunction of the spindle checkpoint regulation, we generatedand expressed fusion proteins of Aurora A or its kinase deadmutant with Hec1 tagged with GFP (GFP-Hec1-AurA andGFP-Hec1-AurA-KD) to target Aurora A to the kinetochores(Fig. 2A and supplemental Movie S3). Furthermore, we treatedHeLa cells by STLC, a specific Eg5 inhibitor that weakens theinteraction of Eg5 with microtubules resulting in the failure ofbipolar spindle formation and mitotic arrest (38, 39), to syn-chronize the cells in prometaphase and then with 200 nM

AZD1152 for 1 h to inhibit their endogenous Aurora B, andimmunostained the cells for the spindle checkpoint proteinBubR1. We observed that AZD1152 treatment abolished thekinetochore localization of this spindle checkpoint protein, andthe chromosomes were misaligned, whereas the DMSO-treated control cells clearly had BubR1 on their kinetochores

(Fig. 2, B and D). Then the cells expressing GFP-CENPB-Au-rora A, GFP-Hec1-Aurora A, GFP-Hec1-Aurora A-KD, andGFP-Aurora A were synchronized to prometaphase by STLCand treated with 200 nM AZD1152 for 1 h. The results showedthat when GFP-Hec1-Aurora A was expressed, the spindlecheckpoint was rescued, as indicated by the localization ofBubR1 on the kinetochores (Fig. 2, C and D). When GFP-Hec1-Aurora A-KD was expressed, BubR1 localization on the kineto-chores was abolished in the presence of AZD1152, indicatingno functions of GFP-Hec1-Aurora A-KD for the localization ofBubR1. Moreover, expressing GFP-CENPB-Aurora A andGFP-Aurora A that could not localize to the kinetochores alsocould not rescue the localization of BubR1 on the kinetochore inthe presence of AZD1152 (Fig. 2, C and D). Thus, the forced local-ization of functional Aurora A on the kinetochores rather than theother places, including the centromere, is sufficient to substituteAurora B for the function of activating the spindle checkpoint.

We further knocked down Aurora B by siRNA to make thefunctional loss defect of Aurora B. The result showed that theAurora B knockdown significantly reduced the localization ofBubR1 on the kinetochores by �70% in comparison with theirrelevant control knockdown (Fig. 2, E and F). By expressingexogenous Aurora B, we performed the rescue experiment andfound that exogenous GFP-Aurora B expression could rescuethe defect by up to 95%. When GFP-Hec1-Aurora B wasexpressed, it could rescue the defect by up to �95% (Fig. 2,E–G). We also expressed GFP-Hec1-Aurora A in Aurora Bknockdown cells. Interestingly, we observed that GFP-Hec1-Aurora A could almost fully rescue the defect caused by AuroraB knockdown (Fig. 2, F and G). In contrast, GFP-CENPB-Au-rora A expression was not able to rescue the defect (Fig. 2, C andD), indicating that the centromere-targeted GFP-CENPB-Au-rora A could not be able to reach the substrates of Aurora B atthe kinetochores. Together, these results demonstrate that thespindle checkpoint defect caused by Aurora B knockdown canbe fully rescued by targeting Aurora A to the kinetochores tosubstitute endogenous Aurora B for regulating the kinetochorelocalization of BubR1.

Centrosome-localized Aurora B Can Substitute Aurora A inPromoting the Mitotic Entry—The active Aurora A at the cen-trosome is required for the timely mitotic entry. In G2 phase,Aurora A directly phosphorylates Cdc25B at Ser-353 and reg-ulates its localization at the centrosome to activate CDK1kinase (40). Aurora A, with its co-activator Bora at the centro-some, also activates Plk1 by phosphorylating it at Thr-210.Then, Plk1, Aurora A, Cdc25B, and Cdk1 form a feedback loopand positively regulate each other’s activity for the mitotic entry(41). To investigate whether Aurora B can substitute Aurora Aon the centrosome to regulate the mitotic entry, we constructeda fusion protein GFP-PLK4CTS-Aurora B, which contains aPLK4 centrosome targeting sequence (CTS, aa 570 – 820) thatmay lead the protein to the centrosomes (42), and expressed itin HeLa cells. We observed that GFP-PLK4CTS-Aurora B wasspecifically localized to the centrosomes both in mitotic andinterphase cells in addition to a fraction of it in the nucleus andthecentromeres(Fig.3AandsupplementalMovieS4).Thephos-phorylation status identification of Aurora B at the T-loop indi-cated that GFP-PLK4CTS-Aurora B was autophosphorylated




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FIGURE 2. Forced kinetochore localization of Aurora A performs the function of Aurora B on the spindle checkpoint regulation. A, immunofluorescencephotomicrographs of HeLa cells transfected with GFP-Aurora A or GFP-Hec1-Aurora A and stained with a CREST antibody. Note that Aurora A (Aura) and itskinase-dead mutant (KD) were localized to the spindle and centrosomes in mitosis, whereas part of Hec1-Aurora A and its relevant kinase-dead mutant werelocalized to the kinetochores. Scale bar, 10 �m. B, HeLa cells were synchronized to prometaphase by STLC and treated by 200 nM AZD1152 for 1 h followed byimmunofluorescence labeling with an anti-BubR1 antibody. Note that inhibiting Aurora B kinase activity resulted in the loss of BubR1 on the kinetochores. Scalebar, 10 �m. C, HeLa cells were transfected with GFP-Hec1-Aurora A, GFP-Hec1-Aurora A-KD, GFP-CENPB-Aurora A, or GFP-Aurora A, synchronized to prometa-phase by STLC and treated with 200 nM AZD1152 for 1 h followed by immunostaining with an anti-BubR1 antibody. Note that expressing GFP-Hec1-Aurora Acould rescue the loss of BubR1 on the kinetochores induced by AZD1152 treatment, whereas GFP-Hec1-Aurora A-KD, GFP-CENPB-Aurora A, and GFP-Aurora Acould not. Scale bar, 10 �m. D, quantitative characterization of HeLa cells treated as in B and C. Bars represented the percentage of the cells with BubR1 onkinetochores. Each data point represents three independent experiments with each measuring 50 cells, and error bars indicate S.D. E, Aurora B knockdownby siRNA resulted in the loss of BubR1 on kinetochores. HeLa cells were transfected with Aurora B siRNA and processed for immunostaining with an anti-BubR1antibody. Scale bar, 10 �m. F, quantitative characterization of HeLa cells treated as in E and G. Bars represent the percentage of the cells with BubR1 onkinetochores. Each data point represents 3 independent experiments with each measuring 50 cells, and error bars indicate S.D. G, BubR1 immunofluorescencelabeling of HeLa cells co-transfected with Aurora B siRNA and GFP-Hec1-Aurora A or RNAi-resistant GFP-Hec1-Aurora B. Note that expression of eitherGFP-Hec1-Aurora B or GFP-Hec1-Aurora B rescued the loss of BubR1 on kinetochores caused by endogenous Aurora B knockdown. Scale bar, 10 �m.




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FIGURE 3. Forced localization of Aurora B to centrosome could perform the function of Aurora A in promoting the mitotic entry. A, immunofluorescencephotomicrographs of HeLa cells transfected with GFP-Aurora B or GFP-PLK4CTS-Aurora B (GFP-PLK4CTSAurB) and stained with a �-tubulin (�-tub) antibody. Notethat GFP-PLK4CTS-Aurora B was localized to the centrosomes, whereas GFP-Aurora B was localized to the nucleus and the centromere in interphase and mitosis.Scale bar, 10 �m. B, HeLa cells transfected with GFP-PLK4CTS-Aurora B were synchronized to mitosis and treated with different concentrations of Aurora Ainhibitor MLN8237 for 1 h. Cell extracts were subjected to immunoblot analysis with antibodies to phospho-Aurora A/Aurora B/Aurora-C and Aurora A. Notethat the activity of Aurora A was inhibited by MLN8237 at 50 nM, whereas the activity of GFP-PLK4CTS-Aurora B was not affected. C, quantitative characterizationof mitotic index of the cells expressed GFP-Aurora B, GFP-PLK4CTS-Aurora B, or GFP-PLK4CTS-Aurora B-KD. HeLa cells were synchronized to G1-S phase bythymidine block and released for 8 –13 h. Before immunofluorescence analysis, 50 nM MLN8237 was added for 1 h. DMSO was used as solvent control. Each datapoint represents three independent experiments with each measuring 50 cells, and error bars indicate S.D. D and E, immunofluorescence staining andquantitative characterization of cyclin B1 nuclear localization in HeLa cells expressed GFP-Aurora B, GFP-PLK4CTS-Aurora B, or GFP-PLK4CTS-Aurora B-KD. HeLacells were synchronized and treated with MLN8237 as in C and stained with cyclin B1 antibody. Cyclin B1 nuclear localization was defined by its staining andis stronger in the nucleus than in the cytoplasm. Each data point represents three independent experiments with each measuring 50 cells, and error barsindicate S.D. Scale bar, 10 �m. F, quantitative characterizations of time of mitotic entry. Cells expressed GFP-Aurora B, GFP-PLK4CTS-Aurora B, or GFP-PLK4CTS-Aurora B-KD were synchronized to G1-S phase by thymidine block and released followed by live cell imaging to measure the duration from the time point ofG1-S phase release to nuclear envelope breakdown. n � 50 cells per group. ***, p � 0.001. Error bars indicate S.D.




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and activated (Fig. 3B). The cells expressing the GFP-PLK4CTS-Aurora B fusion proteins were treated with the Aurora A-spe-cific inhibitor MLN8237 to eliminate the kinase activity ofendogenous Aurora A. The result showed that, in the presenceof MLN8237 at the final concentration of 50 nM and above, theendogenous Aurora A kinase activity was totally inhibited, indi-cated by the phosphorylation of its T-loop, but the kinase activ-ity of GFP-PLK4CTS-AurB was not affected (Fig. 3B). Then thecells expressing the fusion proteins were synchronized to G1-Sphase and released for 8 –13 h followed by the drug treatment.It was found that, in DMSO-treated control cells, the mitoticindex peaked at 10 h after the release, whereas the mitotic indexof MLN8237-treated cells reached the peak level at 12 h (Fig.3C). These indicate that inhibiting Aurora A kinase activitydelayed the mitotic entry. Interestingly, when the cells express-ing GFP-PLK4CTS-Aurora B were treated with MLN8237, themitotic delay by MLN8237 was largely rescued. In contrast, the ki-nase dead mutant GFP-PLK4CTS-Aurora B-KD, that had thesimilar centrosome localization and GFP-Aurora B, failed toalleviate the mitotic delay by MLN8237 treatment. In the cellsexpressing the fusion proteins and treated with MLN8237 wealso checked the nuclear translocation of cyclin B1 that pre-cedes the nuclear envelope breakdown and is required for theG2-M transition (43). Consistent with the mitotic index data(Fig. 3C), the timing of cyclin B1 nuclear translocation inMLN8237-treated cells was largely delayed, and the delay couldbe rescued by expressing GFP-PLK4CTS-Aurora B but not GFP-PLK4CTS-Aurora B-KD or GFP-Aurora B (Fig. 3, D and E). Fur-thermore, we directly determined the time of mitotic entry byexamining the duration from the time point of G1-S phase releaseto nuclear envelope breakdown in cells expressing the fusion pro-teins and treated with MLN8237. Consistent with the resultsabove, we observed that GFP-PLK4CTS-Aurora B indeed couldrescue the mitotic delay (Fig. 3F). Together, these results demon-strate that when Aurora B is artificially forced to locate to thecentrosomes, it can substitute Aurora A to regulate the mitoticentry.

Both N Termini of Aurora A and B Contribute to Their Spa-tial Localizations—It has been reported that the criticaldomains for localization and function of Aurora A and B residewithin their C termini (2, 4), and that the C termini alone couldlocalize to the equivalent sites of and function as the full-lengthAurora A and B(44); however, the functions of the N terminiremain unknown. To determine whether the N termini con-tribute to their localizations and functions, we constructed aseries of GFP-tagged truncation mutants of the human Aurorasand examined their localizations (Fig. 4A). The result showedthat GFP-tagged truncates Aurora A1–121 (GFP-Aurora A aa1–121) showed a diffuse pattern with a significant fraction onthe spindle and spindle poles in HeLa cells, in comparison withthe centrosomal localization of Xenopus Aurora A N-terminaltruncate in Xenopus cells XL2 and egg extracts (45) and thatAurora B1– 65 (GFP-Aurora B aa 1– 65) was largely in thenucleus in G2/prophase and smeared in the cytoplasm but notin the centromere in mitosis (Fig. 4, B and C, and supplementalMovies S5 and S6). We also observed that the catalytic C-ter-minal truncates GFP-Aurora A aa 123-end and GFP-Aurora B66-end showed the similar localizations with the wild-type

Aurora A and B in mitosis, respectively (Fig. 4D), as reported(46). However, when the N terminus of Aurora A was substi-tuted by the N terminus of Aurora B to construct a chimeraprotein GFP-Aurora B-A and expressed in cells, this chimeraprotein was found to be largely localized to the nucleus likeAurora B in G2/prophase and relocated to the area of the wild-type Aurora A in mitosis (Fig. 4, E and F, and supplementalMovie S7), indicating that the N terminus of Aurora B maycontribute to its nuclear localization before the mitotic entry. Incomparison, we fused the N terminus of Aurora A with the Cterminus of Aurora B (GFP-Aurora A-B) and expressed it incells. Surprisingly, we observed that this chimera proteinshowed the centrosomal localization in G2/prophase likeAurora A, and this localization was enhanced before the mitoticentry followed by a typical localization of Aurora B on the cen-tromere and midzone/midbody in mitosis (Fig. 4, D and G, andsupplemental Movie S8). Consistently, we also noticed thatAurora A depleted of its N terminus showed the reduced cen-trosomal localization and Aurora B depleted of its N terminusshowed the reduced nuclear concentration (Fig. 4, D, F, and G).These indicate that the N terminus of Aurora A and B maycontribute to their respective localization before the mitoticentry. Together, these results demonstrate that the divergent Ntermini of the Auroras may contribute to their localizations ininterphase and that once the cell enters mitosis, the C terminimay mainly determine the canonical localizations of the AuroraA and B proteins.

Next, we set to test the functions of the chimera proteins. Byknocking down endogenous Aurora B and expressing GFP-tagged exogenous Aurora B, Aurora A, or Aurora B-A, wefound that both exogenous Aurora B and Aurora B-A, but notAurora A, could efficiently rescue the phosphorylation defect ofhistone H3 at Ser-10 in the nucleus caused by endogenousAurora B knockdown (Fig. 4, H and I). This indicates that the Nterminus of Aurora B may contribute its localization and func-tion. By knocking down endogenous Aurora A and expressingGFP-tagged exogenous Aurora A, Aurora B, or Aurora A-B, wetested the function of the Aurora A N terminus through exam-ining the mitotic index. We observed that the mitotic entry wasdelayed when Aurora A was knocked down and the delay couldbe rescued by GFP-Aurora A and GFP-Aurora A-B but notGFP-Aurora B (Fig. 4J). Furthermore, we measured the dura-tion from the time point of G1-S phase release to nuclear enve-lope breakdown in these cells. Consistent with previous result,we found that, like GFP-Aurora A, GFP-Aurora A-B also res-cues mitotic delay, indicating that the N terminus of Aurora Atargeted the chimera protein GFP-Aurora A-B to the typicallocalization site of Aurora A, where kinase activity rescued thedefect of Aurora A knockdown (Fig. 4K). Collectively, we con-clude that the N termini of both Aurora A and B contribute totheir localizations before mitosis and hence functions of theboth kinases.

N-terminal Deletion of Aurora A Resulted in Prolonged Mito-sis and Defect in Spindle Bipolarity—Next, we set to furtherstudy the roles of the N terminus of Aurora A by depletingendogenous Aurora A and rescuing it with different constructs.First, we knocked down Aurora A in HeLa cells that stablyexpressed GFP-�-tubulin and observed by time-lapse micros-




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copy that, in comparison with the control cells, the spindleassembly in Aurora A knockdown cells was abnormal withextra poles, and the mitosis was prolonged (Fig. 5, A–D), con-sisting with previously reports (47, 48). Then, we depletedAurora A from HeLa cells and transiently expressed full-lengthGFP-Aurora A. We observed that the cells were able to formnormal bipolar spindle and divided normally. In contrast, the Cterminus of Aurora A (GFP-Aurora A aa 123-end) could notefficiently rescue the defects caused by endogenous Aurora A

knockdown (Fig. 5, C, E, and F). The cells assembled extra poles,the chromosomal congression was defective, the mitosis lasted sig-nificantly longer, and the cell division was abnormal (Fig. 5F).Taken together, we conclude that the N terminus contributes tothe function of Aurora A in regulating the spindle bipolarity.

N-terminal Deletion of Aurora B Results in Reduced Phosphor-ylation of Hec1 and Abnormal Mitosis—We also further studiedthe function of the N terminus of Aurora B with similar knock-down and rescue experiments. When Aurora B was depleted

FIGURE 4. N termini of both Aurora A and B contribute to their spatial localizations and functions. A, schematic depiction of the truncate and chimeraprotein constructs of N and C termini of Aurora A (AurA) and B (AurB) tagged with GFP. GFP-Aurora A-B is composed of the N terminus of Aurora A and Cterminus of Aurora B. Conversely, GFP-Aurora B-A is composed of the N terminus of Aurora B and C terminus of Aurora A. B, localizations of the wild-typefull-length GFP-Aurora A and GFP-Aurora B in the cell cycle. Scale bar, 10 �m. C, localizations of the N-terminal truncates of Aurora A and B in the cell cycle. Scalebar, 10 �m. D, localizations of the C-terminal truncates of Aurora A and B in the cell cycle. Note the reduced localization of the C terminus of Aurora A on thecentrosome in interphase cells compared with the full-length wild-type Aurora A in B. Scale bar, 10 �m. E, localization of the chimera proteins GFP-Aurora A-Band GFP-Aurora B-A in the cell cycle. Note that GFP-Aurora A-B was localized on the centrosomes in interphase cells like wild-type Aurora A and in thecentromeres of mitotic cells like wild-type Aurora B, whereas GFP-Aurora B-A was concentrated in late interphase nucleus like wild-type of Aurora B and on thespindle/poles in mitosis like wild-type Aurora A. Scale bar, 10 �m. F, quantitative characterizations of the nuclear accumulation of GFP-Aurora B, GFP-Aurora B66-end, GFP-Aurora B-A, and GFP-Aurora A. Nuclear accumulation was defined by its stronger staining in the nucleus than in the cytoplasm. G, quantitativecharacterizations of the centrosome localization of GFP-Aurora A, GFP-Aurora A 123-end, GFP-Aurora A-B, and GFP-Aurora B. Each data point represents 3independent experiments with each measuring 100 cells, and error bars indicate S.D. H, immunofluorescence photomicrographs of HeLa cells co-transfectedwith Aurora B siRNA and GFP-Aurora A, RNAi-resistant GFP-Aurora B, or GFP-Aurora B-A and stained with anti-H3pS10 antibody. Scale bar, 10 �m. I, quantitativecharacterization of HeLa cells treated as in H. Bars represent the percentage of the cells with histone H3 Ser-10 phosphorylation. Each data point representsthree independent experiments with each measuring 200 cells, and error bars indicate S.D. J, quantitative characterization of mitotic index of HeLa cellsco-transfected with Aurora A siRNA and RNAi resistant GFP-Aurora A, GFP-Aurora B, or GFP-Aurora A-B. The cells were synchronized to G1-S phase by thymidineblock and released for 8 –13 h. Each data point represents three independent experiments with each measuring 100 cells, and error bars indicate S.D. K,quantitative characterizations of the time of the mitotic entry. Cells treated as in J were released from G1-S followed by live cell imaging to measure the durationfrom the time point of G1-S phase release to nuclear envelope breakdown. n � 50 cells per group; ***, p � 0.001. Error bars indicate S.D. ns, not significant.




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from HeLa cells that stably expressed GFP-�-tubulin, bipolarspindles could form, but the chromosome congression wasslow; the cells were finally committed to exit mitosis after a long

arrest and without successful division (Fig. 6, A–D). These cellscontained either a single large nucleus or several small nuclei.Then we performed the rescue experiments in HeLa cells using

FIGURE 5. N-terminal deletion of Aurora A results in prolonged mitosis and multipolar spindle assembly. A and B, HeLa cells stably expressing GFP-�-tubulinwere transfected with irrelevant (A) or Aurora A (AurA) siRNA (B) and subjected to time-lapse microscopy. RFP-H2B was transiently expressed as a chromatin/chromosome indicator. Note that Aurora A depletion resulted in multipolar cell division. Scale bars, 10 �m. C and D, quantitative characterizations of the duration ofmitosis with live cells (C) and percentage of the cells with abnormal spindle assembly with fixed cells (D) shown in (A, B, E, and F) (see below). *, p � 0.05; **, p � 0.01;***, p � 0.0001. The duration of mitosis is determined from the nuclear envelope breakdown to two daughter cells formation. Each data point represents 3 indepen-dent experiments with each measuring 50 cells, and error bars indicate S.D. E and F, HeLa cells co-transfected with Aurora A siRNA-, RFP-H2B-, and RNAi-resistantGFP-AuroraA(E)orGFP-AuroraAaa123-end(F)wereviewedbytime-lapsemicroscopy.NotethatAuroraAaa123-endcouldnotrescueAuroraAdepletion.Scale bars,10�m.




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either the full-length GFP-Aurora B or the C terminus truncateaa 66-end of GFP-Aurora B. The results showed that the full-length GFP-Aurora B successfully rescued the defect caused byendogenous Aurora B knockdown; in contrast, the N terminus

truncate was less efficient in rescuing these defects, and thechromosome congression still failed leading to one largenucleus or multinuclei formation (Fig. 6, C–F). We furtherexamined the phosphorylation of Hec1 at Ser-55 on the kineto-

FIGURE 6. N-terminal deletion of Aurora B results in reduced phosphorylation of Hec1 on kinetochores and abnormal mitotic exit with formation ofmultinuclei or single large nucleus. A and B, HeLa cells stably expressing GFP-�-tubulin were transfected with irrelevant (A) or Aurora B (AurB) siRNA (B) andsubjected to time-lapse microscopy. RFP-H2B was transiently expressed as a chromatin/chromosome indicator. Note that Aurora B depletion resulted inabnormal mitotic exit with formation of multinuclei or single large nucleus. Scale bars, 10 �m. C and D, quantitative characterizations of the duration of mitosiswith live cells (C) and percentage of the cells with multinuclei or a large nucleus (D). *, p � 0.05; ***, p � 0.0001. Duration of mitosis is determined from thenuclear envelope breakdown to two daughter cells formation. Each data point represents 3 independent experiments with each measuring 50 cells, and errorbars indicate S.D. (E and F) HeLa cells were co-transfected with Aurora B siRNA, RFP-H2B and RNAi resistant GFP-Aurora B (E) or GFP-Aurora B aa 66-end (F) andviewed by time-lapse microscopy. Note that Aurora B aa 66-end could not rescue Aurora B depletion. Scale bars, 10 �m. G, Aurora B knockdown by siRNAresulted in decreased phosphorylation of Hec1 on kinetochores. HeLa cells were transfected with Aurora B siRNA and processed for immunostaining with anantibody to phospho-Hec1-Ser-55. Scale bars, 10 �m. H, HeLa cells co-transfected with Aurora B siRNA and RNAi-resistant GFP-Aurora B or RNAi-resistantGFP-Aurora B aa 66-end and processed for immunostaining with an antibody to phospho-Hec1-Ser-55. Note that expression of GFP-Aurora B aa 66-end couldnot fully rescue the phosphorylation status of Hec1. Scale bars, 10 �m. I, quantitative characterization of HeLa cells treated as in G and H. Bars represent thepercentage of cells with phosphorylated Hec1 on kinetochores. Each data point represents three independent experiments with each measuring 50 cells. Errorbars indicate S.D. *, p � 0.05.




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chores, which is thought to be regulated by Aurora B (42). Weobserved that when Aurora B was knocked down by siRNA, thephosphorylation of Hec1 Ser-55 on the kinetochores was sig-nificantly reduced and that this defect could be rescued by full-length wild-type Aurora B but not the C terminus truncate aa66-end of GFP-Aurora B (Fig. 6, G–I). Together, we concludethat the N terminus is required for the full function of Aurora Bin phosphorylating its substrates on the kinetochores, which isrequired for normal cell division. This also indicates that the Nterminus of Aurora B may play an important role in bringingcentromere located Aurora B to the kinetochores to phosphor-ylate its substrates such as Hec1, DSN1, and KNL1, suggest-ing that N terminus-mediated relocation of Aurora B fromthe centromere to kinetochore is needed for the regulation ofkinetochore-microtubule connections.

Discussion

In this study we reveal the relations between the spatiotem-poral compartmentalizations and functions of Aurora A and Band show that one may carry out the functions of the otherwhen one is artificially localized to the places of the other. Wealso reveal that the divergent N termini of the Auroras haveroles for their spatiotemporal localizations. When the N ter-

mini of Aurora A was fused to the C termini of Aurora B, thechimera proteins could localize and function like Aurora A inaddition to like Aurora B and vice versa. Consistent with theprevious reports, our results have shown that Aurora A and Bhave similar potential substrates and kinase activity, and theirspecific functions are likely determined by their different local-izations. Therefore, the functional divergence of Aurora A andB is likely determined by the spatial compartmentalization oftheir binding partners that binds both the conservative C andthe divergent N termini of the Aurora kinases.

Previously we and others have uncovered that the localiza-tions of both Aurora A and B are mainly determined by theirsubstrates and binding partners, and their functions rely ontheir catalytic C termini (4, 44). We revealed that a single aminoacid change on residue Gly-198 can convert Aurora A intoAurora B-like kinase (4). The mutant Aurora AG198N exhibitsthe Aurora B-like localization on the centromere and midzone,where it interacts with components of the chromosome passen-ger complex and rescues the chromosome misalignmentcaused by Aurora B knockdown. A longer side chain on residue198 of mutant Aurora AG198N prevents its attachment to TPX2,and increased hydrophilicity of residue 198 also promotes

FIGURE 7. Model depicting the evolution process of Aurora kinases. A, the sequence alignment of a segment of the Auroras from various species showingthe residue corresponding to Gly-198 of human Aurora A (Arrow) is divergent in polar Auroras (Aurora A like) and equatorial Auroras (Aurora B like). B, modeldepicting the evolution process of Aurora kinases. Primitive Aurora kinase is bifunctional and localized on both polar and equatorial regions of the cells. Geneduplication and divergence occurred independently in protostome and deuterostome. The divergence on the residue corresponding to Gly-198 of humanAurora A results in differential affinity to TPX2 and INCENP. The divergent N terminus along with the residue corresponding to Gly-198 of human Aurora A ledto spatial differentiation of Aurora A and B and thus conferred further the functional divergence of the modern Auroras.




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Aurora AG198N to interact with INCENP. This indicates thatthe slight difference in structure of Aurora A and B leads todifferential interaction with the binding partners of each otherand thus results in differentiation of localization and function.In this work we further find that the divergent N termini of bothAurora A and B may have their own binding partners and con-tribute to the distinct localizations. Without the N terminus,the ability of Aurora A in regulating the mitotic spindle bipo-larity is impaired, whereas Aurora B cannot fully phosphorylateits substrates on kinetochores and regulate the kinetochore-microtubule connection required for faithful chromosome sep-aration and cell division.

In the evolution process both Aurora A and B may originatefrom a single Aurora protein. Phylogenetic analysis suggeststhat the duplication of Aurora genes has independentlyoccurred in vertebrates, invertebrates, and plants (21, 30). Thepolar Aurora kinases (Aurora A-like) and the equatorial Aurorakinases (Aurora B-like) in invertebrates (such as Caenorhabdi-tis elegans and Drosophila melanogaster) have the similar local-izations and functions with their vertebrate counterpart,although they are not orthologous with the respective verte-brate Aurora A and B. Interestingly, despite confusion in thephylogenetic trees, all polar Aurora kinases in model organismsshare a common feature in that the amino acid sequence cor-responding to human Aurora A residue 198 is glycine or ala-nine, which has a short and hydrophobic side chain (Fig. 7A).On the contrary, all equatorial Aurora kinases share an aminoacid with a long and hydrophilic side chain such as asparagineor threonine at the residue corresponding to human Aurora Aresidue 198. Our previous and present studies together withothers’ have presented that Aurora A and B share great similar-ity in substrates and kinase activity, and the functional diver-gence of Aurora kinases is largely determined by their differentlocalization defined by their binding affinity with specific bind-ing partners. So, it seems that, after the gene duplication eventsin the evolutional process, the mutation on a single amino acidof the Auroras confers the differential interaction affinity withtheir binding partners such as TPX2 and INCENP; and in turn,the Auroras are brought to the polar or equatorial localizationby the differential binding partners, respectively, and thus led tothe differentiation of their functions (Fig. 7B). Furthermore,their elongated and low conservative N termini suggest thatthey may have differential binding sites for their additionalbinding partners that may bring them to their special locationsto perform their distinct functions. To fully understand theevolution and function of the Auroras, it is worthy identifyingthese additional binding partners in the future.

Author Contributions—C. M. Z., Q. J., F. D. Z. and S. L. conceivedand coordinated the study and wrote the paper. S. L., Z. X. D., J. Y. F.,C. Y. X., G. W. X., Z. G. W., J. L., G. W., S. L. Z., and B. Y. Z.designed, performed, and analyzed the experiments.

Acknowledgments—We thank all the other members in our laborato-ries for useful comments.

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ZhangGang Wang, Shuli Zhang, Boyan Zhang, Fangdong Zou, Qing Jiang and Chuanmao Si Li, Zhaoxuan Deng, Jingyan Fu, Caiyue Xu, Guangwei Xin, Zhige Wu, Jia Luo,

Spatial Compartmentalization Specializes the Function of Aurora A and Aurora B

doi: 10.1074/jbc.M115.652453 originally published online May 18, 20152015, 290:17546-17558.J. Biol. Chem.

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Spatial Compartmentalization Specializes the Function of Aurora A